The alanine racemase topology is termed Fold type III and is unique among PLP-containing enzymes. It seems likely, therefore, that designing inhibitors that interact with conserved motifs found in the entryway could Mocetinostat in vivo represent a potential source of specificity in the drug design process. Interfering with active site assembly would, in the case of alanine racemase, require compounds that inhibit dimer formation, none of which have been reported for alanine racemase to date. However, dimer inhibitors have been reported in other systems such as HIV protease [[53–55]]. Finally,

a compound that could enter the active site of alanine racemase then undergo a conformational switch rendering the enzyme inactive would make an effective inhibitor, but this type of inhibitor has not yet been reported for this class of enzyme. Conclusions Alanine racemase is a promising target for antibacterial drugs because it is both essential in bacteria and absent in humans. We report the high-resolution crystal structure of alanine racemase from S. pneumoniae. Overall, the structure shares the conserved active site and topology found across all alanine racemases. Known alanine racemase inhibitors such as D-cycloserine, alanine phosphonate, and other

substrate analogues are not specific, acting on other PLP-containing enzymes such as transaminases, also found in humans [59, 62]. In order to be clinically relevant, new inhibitors of alanine racemase with more specificity need to be developed. This structure is an essential starting point for the design of more specific inhibitors AZD5363 datasheet of alanine racemase in S. pneumoniae. Our investigations have identified three potential areas in the AlrSP structure that could be targeted in a structure-based inhibitor design: the active site, the Histone Methyltransferase inhibitor residues forming the dimer interface, and the active site entryway in particular, since designing a ‘plug’ to fit the funnel shape of this feature is intuitively attractive. Methods Protein

expression, purification and crystallization The expression, purification and crystallization of AlrSP have been described previously [21]. Briefly, the gene encoding AlrSP was cloned into pET17 (Novagen) and the resulting vector transformed into E. coli BL21 Histamine H2 receptor (DE3) pLysS cells (Novagen). Overexpression of AlrSP was induced in a culture of these cells, which were then lysed to extract the protein. The recombinant AlrSP was purified using ammonium sulfate precipitation, anion-exchange chromatography, hydrophobic interaction chromatography, and finally, size-exclusion chromatography. Crystals of AlrSP were grown at 4°C in 1.2 M Na Citrate, 0.1 M MES, pH 7.2, and 10% glycerol (protein concentration 23 mg/ml, drop size 4 μl + 4 μl) using the sitting drop vapor diffusion method, then flash-frozen in liquid N2 for data collection. No additional cryoprotectant was required.